Molecular Epidemiological Survey Of Porcine Epidemic Diarrhea In Some Areas Of Shandong And Genetic Evolutionary Analysis Of S Gene

Porcine epidemic diarrhea（PEDV）is an acute infectious disease caused by Porcine epidemic diarrhea virus（PEDV）.Infected piglets have severe diarrhea with high mortality,which seriously affects the productivity and economic efficiency of pig farms,and the disease has caused serious negative impact on pig farming.However,studies have shown that,in addition to PEDV,a variety of pathogens can cause diarrhea in piglets,and piglets with clinical manifestations of diarrhea are often accompanied by mixed infections,which undoubtedly brings great difficulties to the diagnosis and prevention of porcine epidemic diarrhea.To clarify the infection status and incidence pattern of PEDV in recent years in some areas of Shandong,we conducted a survey and analysis of pig farms with diarrhea in some areas of Shandong from September 2019 to April 2021,and collected a total of 176 porcine diarrhea clinical material,tested for viruses commonly causing pig diarrhea,including PEDV,using RT-PCR/PCR technology,and tested for PEDV-positive Histopathological and immunohistochemical tests were performed on typical cases of PEDV-positive pigs.The results showed that the positive rate of diarrhea viruses in these 176 samples was 47.73%（84/176）,among which 66 samples（37.5%）were singly infected by PEDV,12 samples（6.82%）were singly infected by Transmissible gastroenteritis virus（TGEV）,and Only 7samples（3.98%）were infected by Porcine rotavirus（Po RV）alone,and one column of mixed infection by PEDV and TGEV was found.The study showed that diarrhea occurred in pigs in Shandong Province mainly due to PEDV infection alone.In terms of season of incidence,the season of occurrence of epidemic diarrhea in pigs in Shandong province showed an insignificant trend,occurring throughout the year and gradually expanding in age.Histopathological tests revealed that the duodenal and jejunal intestinal villi of PEDV-positive cases were atrophied,broken and detached,and the subintestinal mucosa was severely congested.Immunohistochemical test results showed that PEDV was mostly present in the mucosal epithelium of the duodenum and jejunum.In order to further investigate the genetic evolution of the prevalent PEDV strains,fulllength sequence determination and analysis of the S genes of the 12 PEDV isolates obtained in this study were performed.The results showed that the full-length S genes of the 12 PEDV strains ranged from 4155 nt to 4164 nt and encoded 1385-1388 amino acids.The results of homology analysis showed that the nucleotide homology（97.2%-99.9%）and amino acid homology（95.9%-99.7%）among the 12 isolates were high;the nucleotide homology and amino acid homology between the isolates and the reference strain were 93.0%-99.1% and90.9%-99.4%,respectively.Among them,compared with the classical vaccine strain CV777,the nucleotide sequence homology was 93.1%-93.9% and amino acid homology was 91.2%-93.0%,indicating that the homology between the current prevalent PEDV strain and the classical vaccine strain in Shandong Province was low.Further analysis showed that the CV777 vaccine strain had four consecutive amino acid（QGVN）insertions at positions 59-62,an asparagine（N）insertion at position 140,and a glycine（G）deletion at position 160 compared with the 12 isolates in this study.Antigenic index prediction analysis of the S protein revealed significant differences in antigenicity between CV777 and the 12 isolates at20 aa to 280 aa.In addition,we collected 2916 blood samples from pig farms suspected of having porcine epidemic diarrhea and performed antibody detection by enzyme linked immunosorbent assay（ELISA）.The results showed that 1513 samples were positive,with a positive rate of 51.89%.The antibody positivity rate varied greatly among different pig groups,with the highest positive rate of 79.22% in piglets,followed by farrowing sows（57.79%）and hind sows（40.13%）,and the lowest positive rate of 19.57% in commercial pigs.In conclusion,this study conducted a systematic investigation and analysis of the molecular epidemiology of PEDV in some areas of Shandong Province,and detected PEDV serum antibodies in some pig herds.The results of the study will further enrich the epidemiological database of PEDV and provide some basis for the scientific prevention and control of the disease.